EpiVax specializes in the development of epitope-driven vaccines by screening whole genomes for candidate vaccine components. In this application, we propose to develop a safe, new smallpox vaccine based on epitopes conserved between the vaccinia virus (VV) and Variola (VAR) 'immunomes' that could be used as both a prophylactic and a therapeutic intervention in the event of a bioterrorist attack. First, peptide sequences that are conserved between the vaccinia virus (W) and Variola (Var) genomes will be screened for immunodominant epitopes by computer-driven (EpiMatrix) analysis and confirmed using T cells from VV-immunized individuals. Second, selected epitopes will then be aligned and cloned into DNA vaccine vectors optimized for strong and sustained expression in vivo. Third, we will vaccinate HLA-transgenic mice with the DNA vaccine constructs, measuring de novo immune responses to the DNA vaccine constructs using intracellular cytokine flow cytometry. EpiVax will be responsible for selecting the VV/Var epitopes and managing the project. The TB/HIV Research Lab at Brown University (subcontractor) will perform binding assays and develop the oligonucleotide multi-epitope constructs consisting of strings of the selected epitopes. Dr. Crowe's laboratory (subcontractor, Vanderbilt University) will perform ELISpot assays. Dr. Weiner (subcontractor, Stellar Chance Laboratories, University of Pennsylvania) will perform the vaccination studies. The final product of Phase I will be an evaluation of the immunogenicity and protective effect of several prototype smallpox vaccine constructs in HLA transgenic mice. In Phase II we would screen additional epitopes, build the final vaccine constructs, optimize the immunogenicity of the final vaccine constructs with adjuvants and delivery vehicles, and address the safety, toxicity and immunogenicity of the constructs in human subjects. [unreadable] [unreadable]